Project Summary/Abstract Antibiotic resistance due to the activity of multidrug resistance (MDR) efflux pumps is one important mechanism of bacterial drug resistance. EmrE is one of the smallest known MDR transporters, and it has become a prototype for proton-coupled antiport. EmrE harnesses proton import to drive polyaromatic cation efflux in E. coli, thus conferring resistance to a broad range of drugs. We have previously performed the first quantitative measurement of conformational exchange between open-in and open-out states in a transporter by NMR. Our newest result, asymmetric protonation of EmrE, directly contradicts the long-accepted single-site alternating access model for coupled antiport of drugs and protons. It demonstrates how much is still unknown about this deceptively simply transporter and suggests that the well-known promiscuity of MDR efflux pumps may even be even greater. This project uses NMR spectroscopy and extensive liposomal transport assays to investigate the detailed molecular mechanism of EmrE activity. NMR offers a unique tool because protein conformational exchange and protonation events can be monitored separately and simultaneously, allowing novel insight into how proton and drug transport are coupled. Our goal is to combine quantitative biophysical data and functional assessment of the pH dependence and substrates properties that define EmrE activity to develop a novel model for proton-coupled antiport. We will also test different hypotheses for how proton- coupled antiport is achieved for such diverse substrates with different affinities and transport rates. The insights we gain will aid future efforts to combat bacterial antibiotic resistance due to MDR efflux.